Calibration of Methylene Blue Dye
with Dilution Process
by
Andrea Lyn M. Andres
BS ENE 2B
November 11, 2022
Abstract
We conducted a quantitative regression analysis to calculate the concentration of the
unknown sample by finding the absorbance of the standards to analyze the relationship between
the two variables.
The primary objective of this experiment is to determine the absorbance of the diluted stock
solution with various measurement of concentration. Blank sample and working standard solutions
were run in UV-Vis Spectrophotometer with a different wavelength at a maximum of 668 nm.
With the use of standard curve method, a set of standards containing a known amount of the
analyte being measured must be prepared individually, rather than all from the same stock solution,
to avoid errors that may affect the calibration process, resulting in incorrect and inaccurate
instrument calibration. With this, will be able to plot the data, interpret and obtain a reliable study
about the preparation of calibration curves.
1. Introduction
One of the most crucial aspects of any analytical method is the calibration of the response
of the specific instrument with regards to the concentration of the desired analyte to be measured
and identified. In analytical chemistry, “calibration” is defined as the process of ensuring that a
scientific method or instrument will produce results that are accurate and precise. Any instrument
used in research must be properly calibrated to guarantee that the data it produces is valid and
accessible to others. This aspect of the quantitative determination of an analyte is imperative to
understanding the interesting and complex relationship between measured values and analytical
quantities. Commonly, the number of reference samples, also known as standards, is usually as
few as two; they include a standard containing a known amount of analyte, and a blank or standard
containing no known amount of analyte. In connection with this, there are two main ways of
calibrating an instrument: the standard addition and the standard curve method, whereas the
working curve method is the most suitable one to utilize for this preparation.
Methylene blue (MB) is categorized as an organic dye (OD) released as effluents after
various industrial activities and is one of the most abundant pollutants in the aquatic environment.
Significantly, because of its potential toxicity, removing MB from wastewater has been a matter
of necessity in recent times. In analytical chemistry, methylene blue is commonly used as a redox
indicator. When in an oxidizing environment, solutions of this substance are blue, but they become
colorless when treated with a reducing agent.
UV-Vis Spectrophotometer, a quantitative technique used to measure how much a
chemical substance absorbs light. A UV-Vis Spectrophotometer is a device that uses the Beer-
Lambert Law to measure absorbance in the UV-Vis region. It measures the intensity of light
passing through a sample solution in a cuvette and compares it to the intensity of the light before
it passes through the sample. An absorbance is determined by measuring the amount of light with
a particular wavelength that a given substance prevents from passing through it.
2. Methodology
2.1 Preparation of concentrated stock solution
In this experiment, an analytical grade of Methylene Blue dye (MB) was used in the preparation
of calibration curves. Stock solution of methylene blue dye was prepared(1000mg/L) dissolving
required quantity of dye in distilled water to obtain different concentrations via dilution process
(10ppm, 20ppm, 30ppm, 40ppm, 50ppm) of MB dye was prepared by diluting stock solution.
The methylene blue dye was prepared and calibrated in order to analyze the known
concentration of MB using UV-Vis Spectrophotometer at 230nm, 400nm, and 668nm.
2.2 Preparation of standard solution
After preparing the concentrated stock solution, transfer it to a volumetric flask with solvent.
Label each volumetric flask according to different known concentration (10ppm, 20ppm, 30ppm,
40ppm, 50ppm). Pipette the required volume of methylene blue dye into the first flask, note that it
is measured based on its lower meniscus. Add the required volume of solvent(water) to the same
flask, then mix a total amount of 100 ml diluted standard solution to be analyzed. A minimum of
five standards are recommended for a good calibration curve.
2.3 Preparation of calibration curve
With use of UV-Vis Spectrophotometer, we are going to analyze the samples and unknowns.
Transfer the standard solution and blank or unknown sample to cuvettes. The unknown samples
should contain the same buffer and pH as the standards. Place each standard in the UV-Vis
Spectrophotometer and obtain three readings (230nm, 400nm, and 668nm) for each standard and
record the absorbance data in a spreadsheet to establish five point’s calibration curves.
2.4 Graph the values of absorbance and known concentration
Using Microsoft Excel, plot the data with absorbance on the y-axis and concentration on
the x- axis and determine the equation and R-squared of your graph. Examine the plot, the
calibration curve should look linear and have a part that is non-linear, this is referred to as the limit
of linearity.
3. Results and Discussion
Absorbance
Concentration 230nm 400nm 668nm
10 -0.075 0.015 1.428
20 0.316 0.039 1.696
30 -0.129 0.065 1.46
40 0.283 0.094 1.874
50 0.565 0.102 2.871
Table 1. Values of absorbance and known concentration
In the table 1, it shows the findings obtained from three distinctive bonds at 230nm, 400nm,
and 668nm used to analyzed in the UV-Vis Spectrophotometer.
3.5
230nm
3
2.5 400nm
y = 0.0306x + 0.9466
Absorbance(nm)
R² = 0.6727 668nm
2
1.5
1 y = 0.0125x - 0.1821
R² = 0.4613
0.5
y = 0.0023x - 0.0057
0 R² = 0.9773
0 10 20 30 40 50 60
-0.5
Concentration(nm)
Figure 1. Calibration curve of methylene blue dye
In the figure 1, the absorbance is plotted on the x-axis and the concentration is plotted on the
y-axis of the graph. There three readings to be analyze. The slope of the standard curve indicates
how much light if passing through a solution(transmittance) or how much light is being absorbed
by a solution(absorbance). A correlation coefficient close to unity r=1 is considered sufficient
evidence to conclude that the calibration curve is linear.
For the 668nm, it shows that the straight line to meet any points plotted which indicates that
the relation of two variables is weak. For the 400nm, it is the best fit straight line since r value is
close to 1. This indicates a strong positive relationship of the two variables. For the 230nm, it
shows the least linear regression among the three readings where R² = 0.4613. Among the three
readings, 400nm resulted in the best accuracy and precision.
Use the equation y=mx+b of the standard curve and the absorbance value to determine the
actual concentration of your solution. Evaluate the quality of the standard curve above by using
the R² value then multiply the concentration by the dilution factor for each sample.
4. Conclusion
At the end of the experiment, we can conclude that we can identify the relationship between
the absorbance and concentration at different level of wavelength. One factor that influences the
absorbance of a sample is the concentration. As the concentration goes up, more radiation is
absorbed, thus the absorbance rises. Therefore, absorbance is directly proportional to the
concentration. With the use Beer-Lambert law, we can also determine if there is a linear
relationship between the concentration and the absorbance of the solution, which enables the
concentration of a solution to be calculated by measuring its absorbance.
5. References
A level Chemistry. 2013. Calibration. A level Chemistry Uk. Retrieved from
https://alevelchemistry.co.uk/definition/calibration/
Stauffer,M.April 25, 2018. Introductory Chapter: The Many Faces of Calibration and Validation
in Analytical Methodology in the Present Day. CHAPTER METRICS OVERVIEW. Retrieved
from https://www.intechopen.com/
Perez,A. November 11, 2020. Visible Light Spectroscopic Analysis of Methylene Blue in Water;
What Comes after Dimer? ACS Publications. Retrieved from
https://pubs.acs.org/doi/10.1021/acsomega.0c03830
Currie,L. 1998. GUIDELINES FOR CALIBRATION IN ANALYTICAL CHEMISTRY.
INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY. Retrieved from
http://www.cma4ch.org/chemo/ftp/7004x0993.pdf
